The separation of biologically active or important molecules is a key area for biochemistry and molecular biology. One standard method for isolating or identifying such molecules is polyacrylamide-gel electrophoresis (PAGE), for example, where the molecules in question are induced to migrate through a polyacrylamide-gel matrix by an electric field. For a given magnitude of the electric field, the speed at which the molecules migrate is dependent on their mass, charge and conformation where the smaller molecules, or molecules with a high charge/mass ratio migrate at a faster rate than the larger molecules or molecules with a lower charge/mass ratio. After staining of the resultant gel with an appropriate agent able to stain the target molecules, a distribution of bands is revealed in order of molecular mass. Use of molecular markers (a mixture of standard molecules whose masses are known) running in parallel to the sample to be identified or separated allows the molecular masses to be accurately assessed.
Electrophoresis can be performed to separate molecules by properties other than their mass by appropriate treatment of the gel matrix. For example, molecules can be separated by isoelectric focussing, whereby a pH gradient is formed across the gel matrix prior to electrophoresis and molecules are drawn to the point on the gradient where their overall charge is zero (otherwise known as their isoelectric point).
In order to better separate molecules within a sample, it is known to carry out two-dimensional electrophoresis in which a sample is subjected first to an electric field in a first direction and then a second direction. Either the electrophoresis gel will have a property which varies parallel to the first or second direction (e.g. pH) or a treatment will be applied to molecules within the sample, for example a treatment which denatures molecules within the sample, between electrophoresis in the first direction and electrophoresis in the second direction. For example, in order to analyse the composition of a mixture of RNA and DNA molecules of a range of lengths and potentially including both single- and double-stranded molecules, a sample may be separated by electrophoresis in a first dimension, and then denatured to separate double-stranded molecules, and then separated by electrophoresis in a second dimension.
One known way of carrying out two-dimensional gel electrophoresis is to provide a single pair of spaced apart electrodes which are positioned on opposing sides of a region of an electrophoresis gel. Electrophoresis is then carried out in a first direction. After an electrophoresis process, the gel is rotated relative to the electrodes enabling electrophoresis to then be carried out in a second direction.
This process does, however, require manual intervention or complex automation. Accordingly, it has been proposed to provide two-dimensional gel electrophoresis apparatus having two sets of opposed electrodes in situ for carrying out electrophoresis in a first direction (between a first set of opposed electrodes) and then in a second direction (between the other set of opposed electrodes). For example, US 2009/0107841 (Gunnarsson et al.) discloses gel electrophoresis apparatus having two sets of electrodes, arranged such that two electric fields normal to each other can be alternatively applied to the gel without a requirement for the gel to be rotated in between. Gel can be provided in a cassette form enabling rapid separation of mixtures of molecules.
However, in known two-dimensional gel electrophoresis apparatus with two sets of opposed electrodes, each set of opposed electrodes comprises a single elongate electrode extending along one edge of the zone within which electrophoresis can occur. A problem with this arrangement is that because the electrodes are conductors which must remain at substantially the same electrical potential along their entire length, the pair of electrodes which are not in use distort the electrical field generated by a pair of electrodes which are in use, distorting the distribution of sample molecules after electrophoresis and reducing the surface area within which two-dimensional gel electrophoresis can be effectively carried out.
Therefore, some aspects of the current invention aim to provide a two-dimensional gel electrophoresis system in which a more uniform electric field can be produced across the electrophoresis gel in both dimensions to provide a more even distribution of separated molecules and to maximise the useful area of the electrophoresis gel.